Fatty acid nitroalkenes - Multi-target agents for the treatment of sickle cell disease.

Sickle cell disease (SCD) is a hereditary hematological disease with high morbidity and mortality rates worldwide. Despite being monogenic, SCD patients display a plethora of disease-associated complications including anemia, oxidative stress, sterile inflammation, vaso-occlusive crisis-related pain, and vasculopathy, all of which contribute to multiorgan dysfunction and failure. Over the past decade, numerous small molecule drugs, biologics, and gene-based interventions have been evaluated; however, only four disease-modifying drug therapies are presently FDA approved. Barriers regarding effectiveness, accessibility, affordability, tolerance, and compliance of the current polypharmacy-based disease-management approaches are challenging. As such, there is an unmet pharmacological need for safer, more efficacious, and logistically accessible treatment options for SCD patients. Herein, we evaluate the potential of small molecule nitroalkenes such as nitro-fatty acid (NO2-FA) as a therapy for SCD. These agents are electrophilic and exert anti-inflammatory and tissue repair effects through an ability to transiently post-translationally bind to and modify transcription factors, pro-inflammatory enzymes and cell signaling mediators. Preclinical and clinical studies affirm safety of the drug class and a murine model of SCD reveals protection against inflammation, fibrosis, and vascular dysfunction. Despite protective cardiac, renal, pulmonary, and central nervous system effects of nitroalkenes, they have not previously been considered as therapy for SCD. We highlight the pathways targeted by this drug class, which can potentially prevent the end-organ damage associated with SCD and contrast their prospective therapeutic benefits for SCD as opposed to current polypharmacy approaches.

HIC2 controls developmental hemoglobin switching by repressing BCL11A transcription.

The fetal-to-adult switch in hemoglobin production is a model of developmental gene control with relevance to the treatment of hemoglobinopathies. The expression of transcription factor BCL11A, which represses fetal ß-type globin (HBG) genes in adult erythroid cells, is predominantly controlled at the transcriptional level but the underlying mechanism is unclear. We identify HIC2 as a repressor of BCL11A transcription. HIC2 and BCL11A are reciprocally expressed during development. Forced expression of HIC2 in adult erythroid cells inhibits BCL11A transcription and induces HBG expression. HIC2 binds to erythroid BCL11A enhancers to reduce chromatin accessibility and binding of transcription factor GATA1, diminishing enhancer activity and enhancer-promoter contacts. DNA-binding and crystallography studies reveal direct steric hindrance as one mechanism by which HIC2 inhibits GATA1 binding at a critical BCL11A enhancer. Conversely, loss of HIC2 in fetal erythroblasts increases enhancer accessibility, GATA1 binding and BCL11A transcription. HIC2 emerges as an evolutionarily conserved regulator of hemoglobin switching via developmental control of BCL11A.

Identification and characterization of RBM12 as a novel regulator of fetal hemoglobin expression.

The fetal-to-adult hemoglobin transition is clinically relevant because reactivation of fetal hemoglobin (HbF) significantly reduces morbidity and mortality associated with sickle cell disease (SCD) and ß-thalassemia. Most studies on the developmental regulation of the globin genes, including genome-wide genetics screens, have focused on DNA binding proteins, including BCL11A and ZBTB7A/LRF and their cofactors. Our understanding of RNA binding proteins (RBPs) in this process is much more limited. Two RBPs, LIN28B and IGF2BP1, are known posttranscriptional regulators of HbF production, but a global view of RBPs is still lacking. Here, we carried out a CRISPR/Cas9-based screen targeting RBPs harboring RNA methyltransferase and/or RNA recognition motif (RRM) domains and identified RNA binding motif 12 (RBM12) as a novel HbF suppressor. Depletion of RBM12 induced HbF expression and attenuated cell sickling in erythroid cells derived from patients with SCD with minimal detrimental effects on cell maturation. Transcriptome and proteome profiling revealed that RBM12 functions independently of major known HbF regulators. Enhanced cross-linking and immunoprecipitation followed by high-throughput sequencing revealed strong preferential binding of RBM12 to 5' untranslated regions of transcripts, narrowing down the mechanism of RBM12 action. Notably, we pinpointed the first of 5 RRM domains as essential, and, in conjunction with a linker domain, sufficient for RBM12-mediated HbF regulation. Our characterization of RBM12 as a negative regulator of HbF points to an additional regulatory layer of the fetal-to-adult hemoglobin switch and broadens the pool of potential therapeutic targets for SCD and ß-thalassemia.

Cell-Based Therapies for Trabecular Meshwork Regeneration to Treat Glaucoma.

Glaucoma is clinically characterized by elevated intraocular pressure (IOP) that leads to retinal ganglion cell (RGC) and optic nerve damage, and eventually blindness if left untreated. Even in normal pressure glaucoma patients, a reduction of IOP is currently the only effective way to prevent blindness, by either increasing aqueous humor outflow or decreasing aqueous humor production. The trabecular meshwork (TM) and the adjacent Schlemm's canal inner wall play a key role in regulating IOP by providing resistance when aqueous humor drains through the tissue. TM dysfunction seen in glaucoma, through reduced cellularity, abnormal extracellular matrix accumulation, and increased stiffness, contributes to elevated IOP, but current therapies do not target the TM tissue. Stem cell transplantation for regeneration and re-functionalization of damaged TM has shown promise in providing a more direct and effective therapy for glaucoma. In this review, we describe the use of different types of stem cells for TM regeneration in glaucoma models, the mechanisms of regeneration, and the potential for glaucoma treatment using autologous stem cell transplantation.

Alteration of genome folding via contact domain boundary insertion.

Animal chromosomes are partitioned into contact domains. Pathogenic domain disruptions can result from chromosomal rearrangements or perturbation of architectural factors. However, such broad-scale alterations are insufficient to define the minimal requirements for domain formation. Moreover, to what extent domains can be engineered is just beginning to be explored. In an attempt to create contact domains, we inserted a 2-kb DNA sequence underlying a tissue-invariant domain boundary-containing a CTCF-binding site (CBS) and a transcription start site (TSS)-into 16 ectopic loci across 11 chromosomes, and characterized its architectural impact. Depending on local constraints, this fragment variably formed new domains, partitioned existing ones, altered compartmentalization and initiated contacts reflecting chromatin loop extrusion. Deletions of the CBS or the TSS individually or in combination within inserts revealed its distinct contributions to genome folding. Altogether, short DNA insertions can suffice to shape the spatial genome in a manner influenced by chromatin context.

The HRI-regulated transcription factor ATF4 activates BCL11A transcription to silence fetal hemoglobin expression.

Reactivation of fetal hemoglobin remains a critical goal in the treatment of patients with sickle cell disease and ß-thalassemia. Previously, we discovered that silencing of the fetal γ-globin gene requires the erythroid-specific eIF2α kinase heme-regulated inhibitor (HRI), suggesting that HRI might present a pharmacologic target for raising fetal hemoglobin levels. Here, via a CRISPR-Cas9-guided loss-of-function screen in human erythroblasts, we identify transcription factor ATF4, a known HRI-regulated protein, as a novel γ-globin regulator. ATF4 directly stimulates transcription of BCL11A, a repressor of γ-globin transcription, by binding to its enhancer and fostering enhancer-promoter contacts. Notably, HRI-deficient mice display normal Bcl11a levels, suggesting species-selective regulation, which we explain here by demonstrating that the analogous ATF4 motif at the murine Bcl11a enhancer is largely dispensable. Our studies uncover a linear signaling pathway from HRI to ATF4 to BCL11A to γ-globin and illustrate potential limits of murine models of globin gene regulation.

Understanding heterogeneity of fetal hemoglobin induction through comparative analysis of F and A erythroblasts.

Reversing the developmental switch from fetal hemoglobin (HbF, α2γ2) to adult hemoglobin (HbA, α2ß2) is an important therapeutic approach in sickle cell disease (SCD) and ß-thalassemia. In healthy individuals, SCD patients, and patients treated with pharmacologic HbF inducers, HbF is present only in a subset of red blood cells known as F cells. Despite more than 50 years of observations, the cause for this heterocellular HbF expression pattern, even among genetically identical cells, remains unknown. Adult F cells might represent a reversion of a given cell to a fetal-like epigenetic and transcriptional state. Alternatively, isolated transcriptional or posttranscriptional events at the γ-globin genes might underlie heterocellularity. Here, we set out to understand the heterogeneity of HbF activation by developing techniques to purify and profile differentiation stage-matched late erythroblast F cells and non-F cells (A cells) from the human HUDEP2 erythroid cell line and primary human erythroid cultures. Transcriptional and proteomic profiling of these cells demonstrated very few differences between F and A cells at the RNA level either under baseline conditions or after treatment with HbF inducers hydroxyurea or pomalidomide. Surprisingly, we did not find differences in expression of any known HbF regulators, including BCL11A or LRF, that would account for HbF activation. Our analysis shows that F erythroblasts are not significantly different from non-HbF-expressing cells and that the primary differences likely occur at the transcriptional level at the ß-globin locus.

Evolvement of transgenic male-sterility and fertility-restoration system in rice for production of hybrid varieties.

KEY MESSAGE: We have developed a unique male-sterility and fertility-restoration system in rice by combining Brassica napus cysteine-protease gene (BnCysP1) with anther-specific P12 promoter of rice for facilitating production of hybrid varieties. In diverse crop plants, male-sterility has been exploited as a useful approach for production of hybrid varieties to harness the benefits of hybrid vigour. The promoter region of Os12bglu38 gene of rice has been isolated from the developing panicles and was designated as P12. The promoter was fused with gusA reporter gene and was expressed in Arabidopsis and rice systems. Transgenic plants exhibited GUS activity in tapetal cells and pollen of the developing anthers indicating anther/pollen-specific expression of the promoter. For engineering nuclear male sterility, the coding region of Brassica napus cysteine protease1 (BnCysP1) was isolated from developing seeds and fused to P12 promoter. Transgenic rice plants obtained with P12-BnCysP1 failed to produce functional pollen grains. The F1 seeds obtained from BnCysP1 male-sterile plants and untransformed controls showed 1:1 (tolerant:sensitive) ratio when germinated on the MS medium supplemented with phosphinothricin (5 mg/l), confirming that the male sterility has been successfully engineered in rice. For male fertility restoration, transgenic rice plants carrying BnCysP1Si silencing system were developed. The pollination of BnCysP1 male-sterile (female-fertile) plants with BnCysP1Si pollen resulted in normal grain filling. The F1 seeds of BnCysP1 × BnCysP1Si when germinated on the MS basal medium containing PPT (5 mg/l) and hygromycin (70 mg/l) exhibited 1:1 (tolerant:sensitive) ratio and the tolerant plants invariably showed normal grain filling. The overall results clearly suggest that the customized male-sterility & fertility-restoration system can be exploited for quality hybrid seed production in various crops.

Phytochemical analysis of Jatropha curcas L. during different seasons and developmental stages and seedling growth of wheat (Triticum aestivum L) as affected by extracts/leachates of Jatropha curcas L.

Jatropha curcas shows invasive characters and is a significant source of many phytochemicals with varying biological activities. Different plant parts of Jatropha curcas L exhibited variation in their phytochemical constituents. Leaves and ovary walls were found to contain higher contents of total phenols, tannins and phytic acid whereas free amino acids were greater in leaves. Young leaves of Jatropha show greater contents of all these metabolites. Further, plants exhibit seasonal differences as leaves collected during summer (May-June) have greater accumulation of total phenols, tannins and free amino acids however, phytic acid was more during rainy season. Leachates and extracts in their higher concentrations adversely affected the germination and growth of wheat seedlings however, lower concentrations were more or less stimulatory. These treatments not only decreased the length, fresh and dry weight of seedlings but also affected the chlorophyll contents and activity of enzymes such as nitrate reductase, aminotransferases in wheat seedlings however, the activity of superoxide dismutase and ascorbate peroxidases increased. Experiments indicate harmful allelopathic effects of Jatropha leachates /extracts on wheat seedlings, hence further experimentation and analysis is recommended before continued plantation of Jatropha particularly on fertile soils. However. Growth of Jatropha plants on saline soils and their potential for accumulating sodium, potassium and chloride are the attributes suggesting the possibility of use of Jatropha plants in improving saline soils.

Hysteroscopy: a technique for all? Analysis of 5,000 outpatient hysteroscopies.

OBJECTIVE: 1) To investigate the relationship between operator experience and the success of outpatient hysteroscopy; and 2) to determine if the introduction of normal saline and the use of narrow-caliber hysteroscopes and vaginoscopic approach are associated with a lower failure rate. DESIGN: Retrospective study. SETTING: Teaching-hospital based outpatient hysteroscopy clinic. PATIENT(S): Five thousand consecutive women undergoing outpatient hysteroscopy between October 1988 and June 2003. INTERVENTION(S): The hysteroscopies were carried out both by experienced operators and by trainees. Procedures were performed using 4-mm and 2.9-mm telescopes with 5-mm and 3.5-mm diagnostic sheaths, respectively. Between October 1988 and 1996, the uterine cavity was distended with CO(2) (CO(2) period), whereas normal saline was preferred after 1997 (1997-2003: saline period). Traditional technique of hysteroscope insertion and vaginoscopic approach were used depending on operator preference and experience and patient characteristics. MAIN OUTCOME MEASURE(S): Success, failure, and complication rates. RESULT(S): The hysteroscopies were successfully performed in nearly 95% of cases by 362 operators (mean 13.8 hysteroscopies per operator) with different levels of expertise. Failure and complication rates were 5.2% and 5.4%, respectively, without any significant difference between CO(2) and saline periods. Vasovagal attacks and shoulder pain were significantly higher during the CO(2) period. The success of outpatient hysteroscopy was negatively affected by postmenopausal status, nulliparity, need for cervical dilatation or local anaesthesia, traditional technique of hysteroscope insertion, and use of a 5-mm hysteroscope. CONCLUSION(S): A high level of expertise is not a prerequisite to performing hysteroscopy on an outpatient basis. Recent advances in technique and instrumentation facilitate this approach and might encourage greater adoption by the wider gynecology community.

A new technique for temporary ovarian suspension: temporarily displacing the ovaries anterior to the uterus facilitates pelvic side wall access in the laparoscopic treatment of endometriosis.

The adnexa frequently hinder access to the pelvic side wall during laparoscopic surgery for endometriosis. An innovative tactic can facilitate dissection.

Digital recording of surgical procedures using a personal computer.

OBJECTIVE: To develop a system for recording surgical procedure digitally using a personal computer with real-time compression of the video signal. STUDY DESIGN: We built the system around a modern personal computer with a large hard disk to allow recording of over 250 h of continuous surgery. Digital capture from the camera was achieved using a standard external analogue-digital converter linked to the computer via a firewire cable. The software for capturing, compressing and editing movie files were obtained free of charge from the internet. The optimal settings for the software was determined. RESULTS: We have successfully used this system to record over 100 major and minor hysteroscopic, laparoscopic, vaginal and open gynaecological. Despite compression, the quality of the movies was judged to be very good and still images excellent. The recordings could be integrated in to standard presentation. Still pictures could be printed to provide hard copies for patients and medical notes, and movies burnt on to CDs or DVDs. CONCLUSIONS: A digital recording system built around a standard personal computer is relatively cheap, versatile and has a huge capacity to record surgical procedures.

Surgical and radiological management of uterine fibroids--a UK survey of current consultant practice.

BACKGROUND: The aim of this study was to determine the current surgical and radiological management of uterine fibroids by consultants working in the UK. METHODS: A structured questionnaire was posted to all 1439 UK consultants. Non-responders were sent one reminder. The main outcome measures were surgical route and technique used for myomectomy, and the use and availability of uterine artery embolization (UAE). RESULTS: Eight hundred fifty-two (59%) consultants replied. Seven hundred thirty-five (86%) admitted to regular sessions of gynecologic surgery, and 75% of this group performed open myomectomy, 16% laparoscopic myomectomy, and 66% hysteroscopic myomectomy. Open myomectomy: Forty-one percent of consultants performed open surgery on uteri equivalent to 12-week gestational age or less, 87% prescribed preoperative gonadotrophin-releasing hormone agonists (GnRHa) in order to reduce surgical bleeding, with 35% using myomectomy clamps, 23% tourniquets, and 19% vasoconstrictors. Laparoscopic myomectomy: The largest uterine size the majority would attempt was equivalent to a 12-week gestation, 58.6% used preoperative GnRHa, 21% used intraoperative vasoconstrictors, and 1.4% tourniquets in order to minimize bleeding. Hysteroscopic myomectomy: As with laparoscopic myomectomy, the largest uterine size the majority would attempt was equivalent to a 12-week pregnancy. Blood transfusion: Twenty per cent, 10%, and 7% reported the need for blood transfusion in up to 10% of patients undergoing open, laparoscopic, or hysteroscopic myomectomy, respectively. UAE: Fifty-one percent have access to UAE and 40% have referred at least one patient in 2001. CONCLUSIONS: Open and hysteroscopic myomectomy are frequently utilized in contrast to laparoscopic myomectomy. The reported rate of blood transfusion appears low. Although UAE is widely available, the majority of patients are still managed surgically.

A new device for "no touch" biopsy at "no touch" hysteroscopy: the H Pipelle.

A new device for endometrial biopsy at "no touch" hysteroscopy has been developed based on the Pipelle. The modification (H Pipelle) facilitates endometrial sampling after hysteroscopy without the need to insert additional instruments into the vagina.

Chronic ectopic pregnancy diagnosed incidentally in an infertile woman: a case report.

BACKGROUND: Chronic ectopic pregnancy is an enigma. The clinical presentation can be mild, with absent or subtle symptoms. The high incidence of negative pregnancy tests and the poor specificity of sonographic patterns can be misleading, and the correct diagnosis is sometimes established only at surgery or even histopathologically after the operation. We report the first case of a woman who was accidentally diagnosed with chronic ectopic pregnancy during diagnostic laparoscopy performed as part of a routine investigation for primary infertility. CASE: A 28-year-old woman underwent laparoscopyfor infertility. She had a regular menstrual cycle and was asymptomatic. She gave a history of a possible but unconfirmed miscarriage earlier. Her hormone profile was normal apart from a slightly raised prolactin level. An earlier ultrasound showed a polycystic appearance of the ovaries. Laparoscopy was done on the 25th day of the menstrual cycle, and beta-human chorionic gonadotropin was negative. At laparoscopy, a 2-cm mass wasfound in the right fallopian tube. There was no free blood in the pelvis, and no adhesions. Both tubes were patent at hydrotubation. The mass was excised laparoscopically, and histology confirmed a diagnosis of chronic ectopic pregnancy. CONCLUSION: A review of articles on chronic ectopic pregnancy confirmed the difficulty in diagnosing this condition preoperatively.